Mistakes in chromosome segregation during meiosis or mitosis can lead to spontaneous abortion or to abnormalities such as Down syndrome. They have also been implicated in the processes leading to cancer and aging. Our laboratory has undertaken large-scale genetic screens using the organism Drosophila melanogaster to identify genes encoding proteins critical for ensuring proper chromosome segregation. In the first specific aim, we will investigate several novel components of the chromosomal kinetochores that were first found in our screens and that have been conserved among metazoans including humans. One area of focus in this aim is a multisubunit complex including the proteins ZW10, Rod, and Zwilch. This complex targets the molecular motor dynein to the kinetochore, and it is required in a currently unknown fashion for the operation of the spindle assembly checkpoint that regulates anaphase onset. We propose experiments to dissect the structure of this complex and to investigate its interactions with NudE, a dynein-associated protein whose role is currently not well understood. A second area of focus in this aim will explore the possibility that two new proteins identified in our genetic screens may together constitute a new subassembly at the kinetochore also needed for proper chromosome segregation. The second specific aim describes a new area of research for our laboratory. Mutations in greatwall, one of the Drosophila genes found in our screens, cause chromosome undercondensation because of delayed transit through prophase. The greatwall gene encodes a novel protein kinase with an unusual structure that is conserved in insects and vertebrates. We made antibody against the Xenopus Greatwall protein and used it to. deplete this kinase from oocyte extracts. To our surprise, Greatwall depletion prevented the extracts from entering or maintaining M phase. In the second specific aim, we will follow up these intriguing observations in order to understand Greatwall's role in mitotic progression. We propose experiments to define Greatwall's substrates as well as the upstream kinases involved in Greatwall activation during mitosis. Additional experiments will investigate Greatwall's possible developmental role in oocyte maturation and will search for other proteins with which this kinase associates. The proposed studies on Greatwall have the potential to offer unique mechanistic insights concerning mitotic entry in vertebrate cells.